Therapeutic agent of uremia containing alarin as the main ingredient

ABSTRACT

The present invention relates to a novel use of alarin derived from a splice variant of galanin-like peptide (GALP) RNA and thus having an amino acid sequence similar to that of galanin, with a therapeutic agent of uremia, and the therapeutic agents of uremia according to the present invention include alarin as the main ingredient.

TECHNICAL FIELD

The present invention relates to a therapeutic agent of uremiacontaining alarin as the main ingredient, and more particularly, to theuse of alarin as a therapeutic agent of uremia with a new use of alarinderived from a splice variant of galanin-like peptide (GALP) RNA andhaving an amino acid sequence similar to galanin.

BACKGROUND ART

Despite of the development of medical technology, there are no effectivemethod of treatments for many diseases yet, and such intractablediseases may include uremia. Uremia refers to a syndrome that showsvarious clinical symptoms resulting from abnormal expression forfunctions of various organs and tissues caused by harmful components ofurine accumulated in blood, which are occurred when the urine cannot bedischarged outside the body due to the breakdown of kidney functionwhatever the underlying disease. The clinical symptoms of uremiainclude, for example, digestive system abnormalities such as anorexia,nausea, vomiting, bad breath, stomatitis, enteritis, nervous systemabnormalities such as indifference, indifference, decreased alertness,depressive state, nausea, lethargy, circulatory system abnomial such asanemia, red blood cell hematopoietic disorder, hypertension, ischemicheart disease, pericarditis, myocarditis and the like, and skin problemssuch as pigmentation, pruritus, subcutaneous bleeding, and uremia iscaused by the urea toxin substance accumulated in the blood. There arevarious urea toxin substances, for example, methyl guanidine, indolecompounds, malondialdehyde, creatinine, β-aminoisolactic add,transketolase inhibitors, polyamines, urea, phenols, and the like.However, it is still unclear which of these urea substances areintrinsically involved in the development of uremic symptoms, and it isunknown whether uresis occurs only by a single substance or by a complexaction of these plural substances. However, it is reported that thelatter is most likely. These facts show clearly the difficulty oftreating uremia with medicine. Whatever its development of occurrence, aperson with uremia can face a fatal situation that leads to death,unless the urinary substance is released out of the body or theconcentration of the urea in the blood is reduced by appropriate means.Up to date, therapies that are effective in the treatment of uremiainclude renal transplantation, dialysis and activated charcoal agenttherapies, and the benefits of the latter two common treatments is thata plurality of urea toxin substances, including unknown materials byphysical means can be removed or diluted nonspecifically.

However, activated charcoal agents are used as internal medicines, andthe mechanism of action of activated charcoal agent is by adsorption andin vitro excretion of various toxic substances generated in thedigestive organs. Thus, activated charcoal agent therapy is referred toas an internal medical agent, but like dialysis therapy it can also beregarded as a physical therapeutic means. In addition to that theactivated charcoal agent is limited for therapeutic effect, there areseveral problems such as difficulty in taking them and a tendency tocause constipation, and thus it are not very satisfactory treatments.

On the other hand, dialysis is the most effective treatment for uremia,and in fact, when discharging the urea toxin substance in vitro bydialysis therapy, there is an advantage that the death of the patientdue to the urea substance can be avoided even if the kidney function isabolished. However, complications caused by dialysis are becoming a newproblem. These include, for example, the development of anemia,nephrotic dystrophy, kidney cancer, fibrinous pericarditis, aluminumosteopathy, amyloidosis, crystalline arthritis, multiple cysts, and thelike as complications due to organ dialysis. In addition, there is aproblem in the medical economy such as the low quality of the patient'ssocial life and the occurrence of high treatment costs that should visitfrequently in the dialysis facility.

There has also been reported a method called “in vivo dialysis” in whichnon-proteinaceous nitrogen but not any physical method is transferredinto the intestinal tract. It is the phenomenon that, when the15-keto-16-halogen-prostaglandin E2 class is administered to the renalfailure model rats, the blood serum creatinine (Cre) and blood ureanitrogen (BUN) are transferred to the organ together with the moistureby its

enteropulling action (action which store a moisture in the digestivetract)

. However, the enteropulling action is an action of storing a moisturein the digestive tract, and therefore, it inevitably involves diarrheaand the reporter also mentions this point. Moreover, no matter how muchblood serum Cre or BUN values are lowered, long-term treatment of thisremedy which involves risk of physical depletion and dehydration due todiarrhea is very burdensome for patients with chronic renal failure andthen is hard to be recognized as practically acceptable treatment.

Therefore, there is a demand for the creation of a treatment method ofuremia or a therapeutic agent of uremia that does not have theabove-described problems. To meet these needs, under the title ofpharmaceutical composition useful for treating uremia, Korean patentpublication No. 1989-0001535 (Patent Document 1) describes “thepharmaceutical composition containing a mixture of several salts of thebasic α-amino and α-keto analog of the branched chain essential aminoacid, the composition being characterized in that it is provided in theform of a gastric poorly solubility or gastric interoperability coatingtablet”.

However, the invention disclosed in the above Patent Document 1 stillhas a problem in that it is difficult to provide a satisfactorytreatment effect for uremia, and thus there is still a need for atherapeutic agent having excellent treatment effect of uremia withlong-term use without causing other side effects.

Meanwhile, alarin derived from a splice variant of galanin-like peptide(GALP) RNA is present in vivo by biosynthesis and synthesized withgenetic engineering, which is a peptide having the amino acid sequenceof APAHRSSTFPKVWVTKTERGRQPLRS. Since these alarin shares only 5 aminoacids together with GALP, it cannot activate GALP specific receptors,and neuropeptides have been reported to be expressed in ganglion cellsof human neuroblastoma tumors and may be potential tumor markers. Formore details on such alarin, reference may be made to InternationalPatent Publication No. WO/2006/094973 (Patent Document 2), which areincorporated herein by reference in their entirety.

The above Patent Document 2 only proposes the use of alarin as atherapeutic agent for neurological keratitis, retinol disease, acute andchronic inflammatory and autoimmune diseases, obesity or growthdeficiency, wound healing, uterine fibroids, endometriosis, andanorexia.

Therefore, the present inventors have recognized and studied to solvethe above-mentioned conventional problems, and as a result, we foundthat a peptide that can be simply provided, alarin exerts an excellentimprovement effect in cisplatin-induced uremia, while being the peptidepresent in the human body do not cause side effects of conventionaltherapeutic agents such as gastrointestinal disorders, liver disorders,renal disorders, and diarrhea so that can be used for a long time as atherapeutic agent for treating uremia, so the present invention can becompleted by the said finding.

TECHNICAL PROBLEM

Accordingly, it is a main object of the present invention to solve theseproblems of the prior art on the base of the background of the problemsin the conventional uremic treatments described at the above. Morespecifically, an object of the present invention is to provide atherapeutic agent of uremia that is able to long-term use with providinga satisfactory treatment of uremia as a therapeutic substance that canbe simply provided, while not causing side effects such as anemia,nephrotic dystrophy, endocarditis, aluminum osteopathy, amyloidosis,crystalline arthritis, multiple cysts, and diarrhea, and also incurringqualitative deterioration and high treatment costs in the social life ofthe patient.

Another object of the present invention is to provide a method which canmore easily provide a therapeutic agent of uremia having theabove-described characteristics.

The present invention may also be aimed at achieving, in addition to theabove-mentioned specific objects, other objects that can be readilyderived by those skilled in the art from the said objects and theoverall description of the present specification.

TECHNICAL SOLUTION

In order to achieve the above object, the present inventors haverecognized the problems in the conventional treatment of uremia and havestudied to solve them. As a result, we obtained the experimental datathat the neuropeptide, alarin, which shares only 5 amino acids withGALP, has an excellent improvement effect in cisplatin-induced uremia,and then reveal that it was possible to provide a therapeutic agent ofuremia, which is composed mainly of alanine from the said results, andthus the object of the present invention could be achieved.

The therapeutic agent of uremia according to the present invention forachieving the above object is characterized in that it comprises alarinas a main component.

According to another embodiment of the present invention, the alarin ischaracterized in that it is the human-derived alarin trifluoroacetatesalt having the following amino acid sequence:

H-Ala-Pro-Ala-His-Arg-Ser-Ser-Thr-Phe-Pro-Lys-Trp-Val-Thr-Lys-Thr-Glu-Arg-Gly-Arg-Gln-Pro-Leu-Arg-Ser-OH-trifluoroacetatesalt.

According to another embodiment of the present invention, the alarin hasa molecular weight of 2894.29, characterized in that it has thefollowing molecular formula:

C₁₂₇H₂₀₆N₄₃O₃₈

According to another embodiment of the present invention, thetherapeutic agent of uremia according to the present invention ischaracterized in that it is formulated to be administered from 0.5 mg to10 mg of alarin per an adult of 60 kg body weight.

According to another embodiment of the invention, the formulation ischaracterized in that it is an injectable one.

According to another embodiment of the invention, the formulation ischaracterized in that it is injected by subcutaneous muscle injection.

BRIEF DESCRIPTION OF THE DRAWINGS

The therapeutic agent of uremia containing alarin according to thepresent invention will now be described by way of example only, withreference to the accompanying drawings, in which:

FIG. 1 shows gene splice variants of galanin-like peptides (GALPs)derived from Murine.

FIG. 2 shows the results of comparing the amino acid sequences of alarinobtained from murine, rats, macaques and humans according to a preferredembodiment of the present invention.

FIG. 3 is a graph showing the concentration of blood urea nitrogen (BUN)in the control group and the group administered the therapeutic agent ofthe present invention in Sprague-Dawley rat model having acisplatin-induced uremia according to example of the present invention.

FIG. 4 is a graph showing the concentration of creatinine (CRE) in thecontrol group and the group administered the therapeutic agent of thepresent invention in Sprague-Dawley rat model having a cisplatin-induceduremia according to example of the present invention.

FIG. 5 is a graph showing the histopathological assay results in thecontrol group and the group administered the therapeutic agent of thepresent invention in Sprague-Dawley rat model having a cisplatin-induceduremia according to example of the present invention, wherein one pointis less than 25% of coronary necrosis at the junction site between thecortex and the cerebral cortex, two points are from 25% to 50% ofcoronary necrosis, three points are from 50% to 75% of coronarynecrosis, and 4 points are greater than 75% of coronary necrosis.

FIG. 6 is a graph showing the micrograph of histopathological assayresults in the control group and the group administered the therapeuticagent of the present invention in Sprague-Dawley rat model having acisplatin-induced uremia according to example of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

Hereinafter, preferred embodiments of the present invention will bedescribed in detail.

The therapeutic agent of uremia according to a preferred embodiment ofthe present invention contains alarin as a main component.

According to another preferred embodiment of the present invention,alarin used as a therapeutic agent of uremia of the present inventionmay be human-derived alarin having the following amino acid sequence:

Ala-Pro-Ala-His-Arg-Ser-Ser-Thr-Phe-Pro-Lys-Trp-Val-Thr-Lys-Thr-Glu-Arg-Gly-Arg-Gln-Pro-Leu-Arg-Ser-OH

According to another preferred embodiment of the present invention,alarin used as a therapeutic agent of uremia of the present inventionmay be a pharmaceutically acceptable salt of human-derived alarin havingthe said amino acid sequence.

By the said pharmaceutically acceptable salts are meant salts whichretain the biological effects of the free acids and bases of specificcompounds and are not biologically or otherwise harmful. The compoundsof the present invention may have functional groups that aresufficiently acidic, sufficiently basic, or both, and thus can reactwith any of the number of inorganic or organic bases, and inorganic andorganic acids to produce pharmaceutically acceptable salts. Exemplarypharmaceutically acceptable salts include salts prepared by reaction ofa compound of the invention with an inorganic or organic acid orinorganic base, for example such as salts including trifluoroacetate,sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate,monohydrogenphosphate, dihydrogenphosphate, metaphosphate,pyrophosphate, chloride, bromide, iodide, acetate, propionate,decanoate, caprylate, acrylate, formate, isobutyrate, caproate,heptanoate, propiolate, oxalate, malonate, succinate, cheverate,sebacate, fumarate, maleate, butane-1,4-dioleate, hexain-1,6-dioleate,benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate,hydroxybenzoate, methoxybenzoate, phthalate, sulfonate, xylenesulfonate,phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate,γ-hydroxybutyrate, glycholate, tartrate, methane-sulfonate,propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate andmandelate.

According to another preferred embodiment of the present invention,alarin used as a therapeutic agent of uremia of the present inventionmay be a human-derived alarin trifluoroacetate salt having the followingamino acid sequence particularly:

H-Ala-Pro-Ala-His-Arg-Ser-Ser-Thr-Phe-Pro-Lys-Trp-Val-Thr-Lys-Thr-Glu-Arg-Gly-Arg-Gln-Pro-Leu-Arg-Ser-OH-trifluoroacetatesalt

According to another preferred embodiment of the invention, the saidalarin has a molecular weight of 2894.29 and has the following molecularformula:

C₁₂₇H₂₀₆N₄₃O₃₈

According to another preferred embodiment of the present invention, thesaid alarin may be stored stably at −20±5° C.

According to another preferred embodiment of the present invention, thetherapeutic agent of uremia of the present invention may be administeredfrom 0.5 mg to 10 mg of alarin per adult of 60 kg body weight to providea uremic treatment effect.

Preferably, the therapeutic agent of uremia of the present invention maybe administered from 1.0 mg to 5.0 mg of alarin per adult of 60 kg bodyweight to provide a uremic treatment effect. More preferably, thetherapeutic agent of uremia of the present invention may be administeredfrom 2.0 mg to 5.0 mg of alarin per adult of 60 kg body weight toprovide a uremic treatment effect. Even more preferably, the therapeuticagent of uremia of the present invention may be administered from 3.0 mgto 5.0 mg of alarin per adult of 60 kg body weight to provide a uremictreatment effect. Most preferably, the therapeutic agent of uremia ofthe present invention may be administered from 4.0 mg to 5.0 mg ofalarin per adult of 60 kg body weight to provide a uremic treatmenteffect.

According to another preferred embodiment of the present invention, thesaid therapeutic agent of uremia of the present invention may beformulated into formulations which is intended to be administered 0.5 mgto 10 mg, preferably 1.0 mg to 5.0 mg, more preferably 2.0 mg to 5.0 mg,even more preferably 3.0 mg to 5.0 mg, and most preferably 4.0 mg to 5.0mg of alarin based on an adult of 60 kg body weight to provide a uremictreatment effect.

According to another preferred embodiment of the present invention, thesaid formulation may be preferably, but not particularly limited to, aninjection.

According to another preferred embodiment of the present invention, thesaid formulation may be preferably, but not particularly limited to, aninjectable one injected by subcutaneous muscle injection.

According to another preferred embodiment of the present invention, whenthe therapeutic formulation of the present invention is an injection, asolvent, solubilizing agent or emulsifying agent is used as the carriercomponent, such as water, ethanol, isopropanol, ethyl carbonate, ethylacetate, benzyl alcohol, benzyl benzoate, propylene glycol,1,3-butylglycol oil, glycerol aliphatic esters, polyethylene glycols orfatty acid esters of sorbitan.

Hereinafter, the present invention will be described in more detail withreference to preferred examples, but the present invention is notlimited to these examples.

EXAMPLE

Cisplatin-induced uremia was evaluated for the effect of administrationof test substance on an improvement of uremia in the Sprague-Dawley ratmodel. Here, G1 is normal control group which is administered with anexcipient, G2 is induced control group which is administered with anexcipient, G3 is group which test substance is administered with 10 μg/1mL/head, and G4 is group which test substance is administered with 20μg/mL/head. Here, test substance is alarin (human) trifluoroacetate saltof the present invention. Cisplatin was intraperitoneally administered(IP) and test substance was intravenously administered (IV).

In addition, the test substance (G3-G4) was administered once at 8 hours(Day 0) after cisplatin administration, and in the case of cisplatin(G2-G4), dose of 5 mg/10 mL/kg was administered once at 8 hours (Day 0)before test substance administration.

The intravenous administration was done by fixing an animal into acorrection frame, and slowly injecting at a rate of within 1 mL/minthrough the micro vein using a syringe equipped with 26 gauges needle.The intraperitoneal administration was performed by fixing the animal bya transdormal skin fixation method and intraperitoneally injecting usinga syringe equipped with 26 gauges needle.

Body weights were measured on the day of administering test substance,inducing and necropsy (Day 7). As a result, the weight level of all thetest substance-administered groups measured on the day of necropsyshowed no significant difference from the normal control group.

In addition, blood is collected from the jugular vein of all livinganimals prior to administration of the test substance, beforeadministration of cisplatin, and necropsy, injected into a tubecontaining a coagulation activator, and allowed to coagulate withstanding at room temperature for about 15 minutes, followed bycentrifugation at 3,000 rpm for 10 minutes. Serum obtained from thecentrifugation was tested for blood urea nitrogen (BUN) and creatine(CRE) using a blood biochemical analyzer (7180 Hitachi, Japan). Theblood biochemical test results showed significantly lower BUN and CRElevels in the high dose group of the test substance than the inducedcontrol group.

Finally, animals were inhaled with ether on an necropsy day toanesthetize and euthanize, and kidneys were extracted and fixed in a 10%neutral buffered formalin solution, and the fixed tissues were cut,dehydrated, paraffin embedded and hematoxylin & eosin stained to make aspecimens for histopathological examination after these histologicaltreatment. And then, the obtained specimen is subjected to speculumusing an optical microscope (Olympus BX53, Japan). Histopathologicalexamination showed that the level of lesion in the high dose group oftest substance was significantly lower than that of the control group.

Therefore, when the test substance is administered with a single dose of10 μg/1 mL/head and 20 μg/1 mL/head in a Sprague-Dawley rat model inwhich uremia was induced by administration of cystine under these testconditions, a tendency to improve the blood biochemical level in thegroup of test substance being compared to the control group of excipientwas observed, and in particular, the BUN and CRE levels of the testsubstance of 20 μg/1 mL/head showed a significant difference compared tothe control group of excipient. In addition, the histopathologicalexamination results showed a dose-related change trend and significantchanges in lesion level. Therefore, alarin of the present invention isbelieved to have an effect on the improvement of uremia incisplatin-induced uremia model.

ADVANTAGEOUS EFFECTS

Since the therapeutic agent of uremia containing alarin as the mainingredient of the present invention which is constituted as describedabove is mainly composed of alarin which is a kind of peptide that ispresent in vivo and can be simply synthesized, it can be easily providedand shows an excellent uremic treatment effect but also does not occurside effects such as anemia, nephrotic dystrophy, epicarditis, aluminumosteopathy, amyloidosis, crystalline arthritis, multiple cysticvesicles, diarrhea, and also does not cause qualitative deterioration inthe patient's social life and high incidence of treatment costsaccompanied with treatment, thereby providing an excellent anti-uremictreatment agent that can be used for a long time, and providing anexcellent effect to solve the problems associated with the conventionaltreatment of uremia.

What is claimed is:
 1. The therapeutic agent of uremia characterized inthat the therapeutic agent comprises alarin as a main component.
 2. Thetherapeutic agent of uremia of claim 1, wherein the said alarin ischaracterized in that it is the human-derived alarin trifluoroacetatesalt having the following amino acid sequence:H-Ala-Pro-Ala-His-Arg-Ser-Ser-Thr-Phe-Pro-Lys-Trp-Val-Thr-Lys-Thr-Glu-Arg-Gly-Arg-Gln-Pro-Leu-Arg-Ser-OH-trifluoroacetatesalt.
 3. The therapeutic agent of uremia of claim 1, wherein the saidalarin has a molecular weight of 2894.29, characterized in that it hasthe following molecular formula:C₁₂₇H₂₀₆N₄₃O₃₈
 4. The therapeutic agent of uremia of claim 1, whereinthe said therapeutic agent of uremia is characterized in that it isformulated to be administered from 0.5 mg to 10 mg of alarin per anadult of 60 kg body weight.
 5. The therapeutic agent of uremia of claim4, wherein the said formulation is characterized in that it is aninjectable one.
 6. The therapeutic agent of uremia of claim 4, whereinthe said formulation is characterized in that it is injected bysubcutaneous muscle injection.